Undifferentiated stem cell culture systems

ABSTRACT

The present application discloses methods expanding SCs in an undifferentiated state, the methods comprising incubating undifferentiated SCs in suspension within a culture system comprising basic medium and knockout serum replacement (KOSR). The methods may also be applicable for selective spontaneous or directed differentiation of SCs into a selected population of somatic cells from a culture system of SCs in suspension, the method further comprising incubating said undifferentiated SCs in culture system that support respectively, spontaneous or directed differentiation of SCs into the selected population of somatic cells. The present application also discloses a culture system for expansion of stem cells (SCs) comprising a suspension of undifferentiated stem cells within basic medium and knockout serum replacement (KOSR). The methods and culture system of the invention may be used for large scale production of differentiated cells.

RELATED APPLICATION

This application is a Continuation-in-Part application ofPCT/IL2005/001397 (published as WO06/07037), which derives priority fromU.S. Provisional Application No. 60/639,809. This application herebyincorporates by reference the entire contents of WO06/07037.

FIELD OF THE INVENTION

This invention relates to stem cells (SC), and particularly to methodsand systems for handling and propagating human embryonic stem cells(hESC).

LIST OF RELATED ART

The following is a list of references which are considered to bepertinent for describing the state of the art in the field of theinvention.

-   (1) Reubinoff, B. E., Pera, M. F., Fong, C. Y., Trounson, A. &    Bongso, A. Embryonic stem cell lines from human blastocysts: somatic    differentiation in vitro. Nat Biotechnol 18, 399-404 (2000)-   (2) Amit, M. et al. Clonally derived human embryonic stem cell lines    maintain pluripotency and proliferative potential for prolonged    periods of culture. Dev Biol 227, 271-278 (2000).-   (3) Xu, C. et al. Feeder-free growth of undifferentiated human    embryonic stem cells. Nat Biotechnol 19, 971-974 (2001).-   (4) Amit, M. et al. Human feeder layers for human embryonic stem    cells. Biol Reprod 68, 2150-2156 (2003).-   (5) Richards, M., Fong, C. Y., Chan, W. K., Wong, P. C. & Bongso, A.    Human feeders support prolonged undifferentiated growth of human    inner cell masses and embryonic stem cells. Nat Biotechnol 20,    933-936 (2002).-   (6) Cowan, C. A. et al. Derivation of embryonic stem-cell lines from    human blastocysts. N Engl J Med 350, 1353-1356 (2004).-   (7) Amit, M., Shariki, C., Margulets, V. & Itskovitz-Eldor, J.    Feeder layer- and serum-free culture of human embryonic stem cells.    Biol Reprod 70(3):837-45 (2004).-   (8) Pera, M. F. et al. Regulation of human embryonic stem cell    differentiation by BMP-2 and its antagonist noggin. J Cell Sci 117,    1269-1280 (2004).-   (9) GB 2409208.-   (10) WO04/031343.-   (11) Xu, R. H., et al. Basic FGF and suppression of BMP signaling    sustain undifferentiated proliferation of human ES cells. Nat    Methods. 3, 164-5 (2005).-   (12) Vallier L, et al. Activin/Nodal and FGF pathways cooperate to    maintain pluripotency of human embryonic stem cells. J Cell Sci.    118, 4495-509 (2005).-   (13) WO 06/070370 (Reubinoff, B. et al.).-   (14) Itsykson, P., et al. Derivation of neural precursors from human    embryonic stem cells in the presence of noggin. Mol Cell Neurosci    30(1), 24-36 (2005).-   (15) Yan, Y. et al. Directed differentiation of dopaminergic    neuronal subtypes from human embryonic stem cells. Stem Cells 23(6),    781-90 (2005).-   (16) Ludwig, T. E. et al. Derivation of human embryonic stem cells    in defined conditions. Nat Biotechnol 24(2), 185-87 (2006).-   (17) Kallos, M. S. et al. Large-scale expansion of mammalian neural    stem cells: A review. Med Biol Eng Comput 41(3), 271-82 (2003).-   (18) Cormier, J. T., et al. Expansion of Undifferentiated Murine    Embryonic Stem Cells as Aggregates in Suspension Culture    Bioreactors. Tissue Eng 1, 1 (2006).-   (19) Fok, E. Y. et al. Shear-controlled single-step mouse embryonic    stem cell expansion and embryoid body-based differentiation. Stem    Cells 23(9), 1333-42 (2005).-   (20) Gerecht-Nir, S. et al. Bioreactor cultivation enhances the    efficiency of human embryoid body (hEB) formation and    differentiation. Biotechnol Bioeng 86(5), 493-502 (2004).-   (21) Goldsborough, M. D. et al. Serum-free culture of murine    embryonic stem (ES) cells. Focus 20(1), 8-12 (1998).-   (22) WO 98/30679.-   (23) WO 03/104444.-   (24) Reubinoff B E, Khaner H. Identification and Maintenance of    Neural Progenitors from human ES cells. In Handbook Of Stem Cells,    Volume 2: Embryonic Stem Cells. Lanza R, Gearhart J D, Hogan B L M,    Mckay R D, Melton D A, Pedersen R, Thomson J A West M D (eds).    Academic Press 2004, pages 511-520.-   (25) Klimanskaya I, Chung Y, Becker S, Lu S J, Lanza R. Human    embryonic stem cell lines derived from single blastomeres. Nature.    Mar. 15, 2007 ;446(7133):342.-   (26) D'Amour K A, Bang A G, Eliazer S, Kelly O G, Agulnick A D,    Smart N G, Moorman M A, Kroon E, Carpenter M K, Baetge E E.    Production of pancreatic hormone-expressing endocrine cells from    human embryonic stem cells. Nat Biotechnol. November    2006;24(11):1392-401.-   (27) Yao S, Chen S, Clark J, Hao E. Beattie G M Hayek A and Ding S.    Long-term self-renewal and directed differentiation of human    embryonic stem cells in chemically defined conditions. PNAS 2006;    103: 6907-6912.

BACKGROUND OF THE INVENTION

Stem cells are immature, unspecialized cells that renew themselves forlong periods through cell division. Under certain conditions, they candifferentiate into mature, functional cells. Human embryonic stem cells(hESC) are derived from early surplus human blastocysts¹. Human ES cellsare unique stem cells since they can self-renew infinitely in culture,and since they have a remarkable potential to develop intoextraembryonic lineages as well as all somatic cells and tissues of thehuman body¹.

Given the unique properties of hESC, they are expected to havefar-reaching applications in the areas of basic scientific research,pharmacology, and regenerative medicine. Human ES cell lines can providea powerful in vitro model for the study of the molecular and cellularbiology of early human development, for functional genomics, drugscreening, and discovery. They may serve for toxicology andteratogenicity high throughput screening. Since hESC can self-renewindefinitely and can differentiate into any cell type, they can serve asa renewable, unlimited donor source of functionally maturedifferentiated cells or tissues for transplantation therapy. Inaddition, transplanted genetically-modified hESC can serve as vectors tocarry and express genes in target organs in the course of gene therapy.

While the promise of hESC for basic scientific research pharmacology andregenerative medicine is remarkable, the exploitation of hESC for mostapplications depends upon further development. Improved control of thegrowth of undifferentiated hESC, the development of bulk feeder-freecultures of undifferentiated cells, the development of animal-freeculture systems, and the development of methods and tools which directthe differentiation and generate pure cultures of mature functionalcells of a specific type are required.

At present, a few culture systems are most commonly used to propagateundifferentiated hESC¹⁻³. In the initial culture system that wasdeveloped, undifferentiated hESC are cultured in serum-containing mediumas colonies, upon a layer of fibroblast feeder cells (of mouse¹ or humanorigin^(4, 10)). It is possible to remove all animal products from thisculture system and replace them with those from a human source⁵. It wasfound that in this system the cells are propagated as clumps on a smallscale, which does not allow cloning².

An alternative culture system for use in the proliferation ofundifferentiated growth of hESC comprises a culture matrix comprisingextracellular matrix (ECM) that may be prepared from feeder cells orother sources and a conditioned medium being preconditioned by feedercells. The suggested leading cells in the feeder cells include primarymouse embryonic fibroblasts (PMEF), a mouse embryonic fibroblast cellline (MEF), murine foetal fibroblasts (MFF), human embryonic fibroblasts(HEF), human foetal muscle (HFM), human foetal skin cells (HFS), humanadult skin cells, human foreskin fibroblasts (HFF)⁹, human adultFallopian tubal epithelial cells (HAFT), or human marrow stromal cells(HMSC).

Another alternative culture system that was developed and usedextensively is a serum-free system that includes the knockout (KO)medium supplemented with knockout serum replacement (KOSR) and FGF2.This system allows cloning of undifferentiated hESC, although at a lowefficiency². Undifferentiated cells are cultured as flat colonies andmay be propagated mechanically as small clusters or single cells (byusing trypsin⁶).

Knockout serum replacement (KOSR) (Gibco) is a chemically defined,serum-free culture medium supplement used as a substitute foranimal-based serum in KO-DMEM-based culture systems for propagating stemcells. KOSR can efficiently promote the growth and maintenance ofundifferentiated embryonic stem cells and therefore may replace thesupplementation with fetal bovine serum (FBS)^((21),) KO-DMEM mayreplace traditional DMEM in either FBS- or KOSR-supplementedcultures⁽²¹⁾.

Undifferentiated propagation of adherent colonies of hESCs may beaccomplished with a KO serum-free culture system without the use offeeders by plating and growing the colonies on extracellular matrices(ECM) within a feeder-conditioned KO-DMEM medium supplemented with KOSRand FGF2³. Furthermore, it has been suggested that feeder conditioningmay be replaced by substituting the medium with high concentrations ofFGF2 and noggin¹¹. Alternatively, feeder conditioning was replaced bytransforming growth factor ⊕1 and human LIF (in addition to FGF2) andgrowing the cells on human fibronectin⁷, or by serum-free mediasupplemented with soluble factors including FGF2, activin A,transforming growth factor β1 (TGFβ1), pipecolic acid, GABA, LiCL andculturing the cells on ECM components(16). In another recentpublication, undifferentiated propagation of hESC colonies, in theabsence of feeders, was reported with a chemically defined mediumwithout serum replacer, supplemented with activin or nodal plus FGF2¹².In general, a key limitation of hESC culture systems is that they do notallow the propagation of pure populations of undifferentiated stem cellsand their use typically involves some level of backgrounddifferentiation. The stem cells most commonly follow a default pathwayof differentiation into an epithelial cell type that grows either as amonolayer of flat squamous cells or form cystic structures. Mostprobably, this form of differentiation represents differentiation ofhESC into extraembryonic endoderm⁸.

In these adherent culture systems of colonies, the hESCs are mostcommonly propagated (mechanically and/or by using enzymatic digestion)as clusters, on a small scale. These culture systems arelabor-intensive, highly variable, may contain undefined factors, and donot provide steady-state operating conditions. Most importantly, they donot typically allow for large scale production of standardizedhomogenous undifferentiated hESCs needed for the aforementioned uses.

Suspension culture bioreactors offer several advantages over theconventional use of static monolayer cultures. These systems facilitatethe large-scale expansion of the cells in a homogeneous cultureenvironment, thus decreasing the risk of culture variability. They arealso less labor-intensive to operate and offer the possibility ofcomputer control and monitoring of the culture conditions. Althoughbioreactors have been used to expand neural stem cells⁽¹⁷⁾, mouse EScells⁽¹⁸⁾ and differentiating hESCs within embryoid bodies (EBs)(20),only recently some progress has been made towards the development ofprotocols for the feeder-free expansion of undifferentiated hESCs insuspension systems¹³.

A major obstacle in developing systems for culturing hESCs in suspensionbioreactors was recently overcome when it was demonstrated that hESCsmay be propagated, in the pluripotent, undifferentiated state, asclusters in suspension, using Neurobasal™ medium as the basic medium ofthe culture system⁽¹³⁾, which is supplemented with N2. Neurobasal™medium is a basal medium especially formulated for growth of neuronalcells, and supplemented with either serum (e.g., FBS) or B27 or N2 serumreplacement²³.

SUMMARY OF THE INVENTION

The present invention is based on the surprising finding thatsupplementing Neurobasal™ medium with KO serum replacement (KOSR)instead of with N2 significantly increased level of expansion ofundifferentiated and pluripotent stem cells cultivated in a suspension.Specifically, the results provided hereinbelow show that after 3 weeksthe total number of undifferentiated and pluripotent human embryonicstem cells (hESCs) propagated in NBSR medium reached a population ofapproximately double that of NBN2-cultured hESCs.

Further, it was surprisingly found that the NBSR medium couldeffectively support long-term cultivation of undifferentiatedpluripotent hESCs. The specific, non-limiting examples providedhereinbelow show that the percentage of hESCs expressing markers ofpluripotent stem cells (SSEA4, TRA1-60, and TRA1-81) was high (>95%) andstable after 3.5 weeks and 6.5 weeks of suspension culture.

Furthermore, it was surprisingly found that NBSR medium supplementedwith Nutridoma-CS did not require the addition of laminin.

Thus, in accordance with a first of its aspects, the present inventionprovides a culture system for expansion of stem cells (SCs) comprising asuspension of undifferentiated stem cells within basic medium andknockout serum replacement (KOSR). Preferably, the basic medium isNeurobasal™.

In accordance with a second aspect, the invention provides a method ofexpanding SCs in an undifferentiated state, the method comprisingincubating undifferentiated SCs in suspension in a culture systemcomprising basic medium and knockout serum replacement (KOSR).

In accordance with a third aspect, the invention provides a method ofpromoting directed differentiation of SCs to a selected population ofsomatic cells, the method comprising:

-   -   (a) providing a culture system comprising a suspension of        undifferentiated SCs in accordance with the invention;    -   (b) incubating said undifferentiated SCs in culture system that        support directed differentiation of SCs into the selected        somatic cells.

In accordance with one embodiment, the selected population of somaticcells consists essentially of neural precursor cells. In accordance withanother embodiment the selected population of somatic cells consistsessentially of dopaminergic neuronal cells. Culture systems whichsupport directed differentiation of SC's into a specifically desirabletype of somatic cell, such as neural precursor cells or dopaminergicneuronal cells are well known in the art.

In accordance with a fourth aspect, the invention provides a method ofpromoting spontaneous differentiation of SCs into somatic cells, themethod comprising:

-   -   (a) providing a culture system comprising a suspension of        undifferentiated SCs in accordance with the invention;    -   (b) incubating said undifferentiated SCs in culture system that        support spontaneous differentiation of SCs into the somatic        cells.

The somatic cells in accordance with this aspect of the invention maycomprise cells from the three embryonic germ layers: ectoderm; mesoderm;and endoderm. Thus, the method of the invention for promotingspontaneous differentiation of somatic cells so as to provide apopulation of somatic cells comprising a single cell type as well as amixture of ectodermal, mesodermal and/or endodermal cells

Culture systems which support spontaneous differentiation of SC's intosomatic cells, are well known in the art.

In accordance with a fifth aspect, the invention provides the use ofKOSR for the preparation of a culture system for expanding in suspensionundifferentiated SCs.

BRIEF DESCRIPTION OF THE DRAWINGS

In order to understand the invention and to show how it may be carriedout in practice, a preferred embodiment will now be described, by way ofnon-limiting example only, with reference to the accompanying drawings,in which:

FIG. 1: is a bar graph showing the number of stem cells in a suspensionculture system comprising Neurobasal™ (NB) medium as the basic mediumsupplemented with KOSR (NBSR) or with N2 (NBN2); Cell counts wereperformed 3 weeks after transfer of equal number of clusters ofundifferentiated hESCs into suspension culture within the two mediacompositions.

FIG. 2: is a dark field micrograph of undifferentiated hESCs in asuspension cultures in NB medium supplemented with KOSR;

FIGS. 3A-3E: are FACS analysis images of hESC clusters that werecultured 3 weeks in suspension and dissociated into single cells andshowing that >90% of the cells expressed markers of pluripotency SSEA-4(FIG. 3A), TRA1-60 (FIG. 3B), TRA1-81 (FIG. 3C) and, but not a marker ofearly neural differentiation, PSA-NCAM (FIG. 3D). Summary of twoindependent experiments are presented in the bar graph histograms (FIG.3E);

FIG. 4A-4B are phase contrast image (FIG. 4A) and fluorescence image(FIG. 4B) of colonies of undifferentiated hESCs developed after platingclusters that were cultivated in suspension and then replated on feederswith typical morphology (FIG. 4A), and with cells within the adherentcolonies expressing alkaline phosphatase (FIG. 4B).

FIG. 5: is a bar graph showing that supplementation of NB withNutridoma-CS, FGF2, activin A, ECM components and neutrophins promotelong term propagation of undifferentiated hESC clusters in suspension,as exhibited in corresponding FACS analysis that showed that thepercentage of hESCs expressing SSEA-4, TRA1-60, and TRA1-81 was high(>95%) and stable after 3.5 and 6.5 weeks of suspension culture.

FIG. 6: is a bar graph showing that supplementation of NB Nutridoma-CShowever, without laminin also promotes long term propagation ofundifferentiated hESC clusters in suspension, as exhibited by theexpression of the different markers (% positive cells) in the presenceor absence of laminin (+LAM and −LAM, respectively) and after 3.5 and6.5 weeks of suspension culture.

FIG. 7: is a bar graph showing the number of cells after long termsuspension culture in NBSR supplemented with Nutridoma-CS beingincreased after 6.5 weeks in the absence (−LAM) as compared to thepresence (+LAM) of laminin supplementation.

FIGS. 8A-8C: are phase contrast image (FIG. 8A), indirectimmuno-fluorescence staining image (FIG. 8B) and image of nucleicounterstaining with DAPI (FIG. 8C) showing cells with morphologicalcharacteristics of neurons emanating from the clusters after inductionin DMEM/F12 supplemented with B27, noggin and FGF2 for 2-3 weeks andculturing for a week on laminin coated slides.

FIG. 9: is an immuno-fluorescent image showing that hESCs that werepropagated in suspension could give rise to midbrain dopaminergicneurons co-expressing TH (light gray) and EN-1 (dark gray); The arrowshows a cell which co-expresses EN-1 and TH, after plating the hESCs onlaminin coated slides and culturing in the presence of FGF8 and SHH for2 weeks.

FIG. 10: is an immuno-fluorescent image showing that hESCs that werepropagated in suspension could give rise to mesodermal cells expressinghuman muscle actin (hMA).

FIG. 11: is an immunofluorescent image showing that hESCs that werepropagated in suspension could give rise to endodermal cells expressingSox17.

FIG. 12: is a bar graph showing that NB with Nutridoma-CS, FGF2, activinA, ECM components and neutrophins promotes long-term propagation ofclusters of undifferentiated hESCs in suspension, as exhibited incorresponding FACS analysis, which showed that the percentage of hESCsexpressing TRA1-60 and TRA1-81 was high and stable after 7 and 10 weeksof suspension culture.

FIGS. 13A-13C: are images of histological sections of teratoma tumorsthat developed after inoculation of hESCs, cultivated in suspension for7 weeks, into the testes of NOD/SCID mice. Differentiated progenyrepresenting the three embryonic germ layers, mesoderm (FIG. 13A),ectoderm (FIG. 13B) and endoderm (FIG. 13C) are illustrated.

DETAILED DESCRIPTION OF EXEMPLARY EMBODIMENTS

The invention is described in the following detailed description withreference to culture systems for handling stem cells, preferably humanembryonic stem cells. It should be noted that in addition to the culturesystems discussed in detailed hereinbelow, also encompassed within thepresent invention are uses of specific components described withreference to the culture system in the preparation of such stem cellcultures, as well as to methods of use of the culture system in handlingstem cell cultures and methods of preparing cultured cells.

As used in the specification and claims, the forms “a”, “an” and “the”include singular as well as plural references unless the context clearlydictates otherwise. For example, the term “a stem cell” includes one ormore stem cells, and the term “stem cells” includes one stem cell aswell as more than one stem cell.

As used herein, the term “or” means one or a combination of two or moreof the listed choices.

Further, as used herein, the term “comprising” is intended to mean thatthe methods and culture systems includes the recited elements, but doesnot exclude others. Similarly, “consisting essentially of” is used todefine methods and systems that include the recited elements but excludeother elements that may have an essential significance on thefunctionality of the culture systems of the inventions. For example, aculture system consisting essentially of a basic medium and mediumsupplements will not include or will include only insignificant amounts(amounts that will have an insignificant effect on the propagation ofcells in the culture system) of other substances that have an effect oncells in a culture. Also, a system consisting essentially of theelements as defined herein would not exclude trace contaminants.“Consisting of” shall mean excluding more than trace amounts of otherelements. Embodiments defined by each of these transition terms arewithin the scope of this invention.

Further, all numerical values, e.g., concentration or dose or rangesthereof, are approximations which are varied (+) or (−) by up to 20%, attimes by up to 10%, from the stated values. It is to be understood, evenif not always explicitly stated that all numerical designations arepreceded by the term “about”. It also is to be understood, although notalways explicitly stated, that the reagents described herein are merelyexemplary and that equivalents of such are known in the art.

In its broadest sense, the present invention concerns culture systemsand methods for the maintenance and preferably propagation ofundifferentiated, pluripotent stem cells (SCs) in suspension. Theculture system provided herein has been found to be especially suitablefor large scale and long term maintenance of undifferentiated stemcells.

Glossary

In the following description and claims use will be made, at times, witha variety of terms, and the meaning of such terms as they should beconstrued in accordance with the invention is as follows:

“Stem cells”, as used herein, refers to cells which under suitableconditions are capable of differentiating into other cell types having aparticular, specialized function (i.e., “fully differentiated” cells)while under other suitable conditions are capable of self renewing andremaining in an undifferentiated pluripotential state as detailed below.A “cell” as used herein refers to a single cell as well as to apopulation of (i.e. more than one) cells. The population may be a purepopulation comprising one cell type. Alternatively, the population maycomprise more than one cell type. The stem cells are preferablyhematopoietic stem cells obtained from bone marrow tissue of anindividual at any age or from cord blood of a newborn individual,embryonic stem (ES) cells obtained from the embryonic tissue formedafter gestation (e.g., blastocyst), or embryonic germ (EG) cellsobtained from the genital tissue of a fetus any time during gestation,preferably before 10 weeks of gestation.

“Embryonic stem cell” and “Pluripotent embryonic stem cell”, as usedherein, refer to a cell which can give rise to many differentiated celltypes in an embryo or an adult, including the germ cells (sperm andeggs). This cell type is also referred to as an “ES” cell.

“Cell culture” or “Cultured cell”, as used herein, refer to cells ortissues that are maintained, cultured, cultivated or grown in anartificial, in vitro environment. Included within this term arecontinuous cell lines (e.g. with an immortal phenotype), primary cellcultures, finite cell lines (e.g., non-transformed cells), and any othercell population maintained in vitro. In this connection, a primary cellis a cell which is directly obtained from a tissue or organ of ananimal, including a human, in the absence of culture. Typically, thoughnot necessarily, a primary cell is capable of undergoing ten or fewerpassages in vitro before senescence and/or cessation of proliferation.

“Undifferentiated pluripotential ES cells”, “Pluripotent SC”, and “ESC”,as used herein, refer to precursor cells that have the ability to formany adult cell. Such cells are true cell lines in that they: (i) arecapable of indefinite proliferation in vitro in an undifferentiatedstate; and (ii) are capable of differentiation to derivatives of allthree embryonic germ layers (endoderm, mesoderm, and ectoderm) evenafter prolonged culture. Human ES cells (hES cells) are derived fromfertilized embryos that are less than one week old (in the cleavage orblastocyte stage) or produced by artificial means (such as by nucleartransfer) that have equivalent characteristics.

“Undifferentiated”, as used herein, refers to cultured cells when asubstantial proportion (at least 20%, and possibly over 50% or 80%) ofthe cells and their derivatives in the population display morphologicalcharacteristics of undifferentiated cells, distinguishing them fromdifferentiated cells of embryo or adult origin. Cells are recognized asproliferating in an undifferentiated state when they go through at least1 population doubling during a cultivation period of at least 3 weeks,while retaining at least about 50%, or the same proportion of cellsbearing characteristic markers or morphological characteristics ofundifferentiated cells after said cultivation period.

“Maintenance” means continued survival of a cell or population of cells,at times, with an increase in numbers of cells. “Proliferation”,“propagation”, “expansion” and “growth”, which may be usedinterchangeably with each other, refer to such an increase in cellnumber. According to one embodiment, this term refers to a continuoussurvival of the cells for at least 6 weeks, preferably for at least 10weeks.

“Cell suspension” as used herein, refers to a culture of cells in whichthe majority of the cells freely float in the medium, typically aculture medium (system), and the cells floating as single cells, as cellclusters and/or as cell aggregates. In other words, the cells surviveand propagate in the medium without being attached to a substrate.

“Culture system”, as used herein, refers to a culture system suitablefor the propagation of SCs. The term denotes a combination of elements,at minimum including a basic medium (a cell culture medium usuallycomprising a defined base solution, which includes salts, sugars andamino acids) and the serum replacement supplement KOSR. The culturesystem in accordance with the invention may further comprise otherelements such as, without being limited thereto, an extracellular matrix(ECM) component, a serum or serum replacement (in addition to KOSR), aculture (nutrient) medium and other exogenously added factors, whichtogether provide suitable conditions that support SC growth. Theconditions are such that SC can proceed through the cell cycle, grow anddivide. Preferably, the conditions are such which enable growth of humanstem cells, preferably, human embryonic stem cells (hESC). Further, theculture system provides conditions that permit the SC to stablyproliferate in the culture system for at least 6 weeks. It is intendedthat the definition encompass outgrowth as well as maintenance media.

“Large scale”, as used herein with regard to cell cultivation andexpansion, refers to the cultivation of SC under conditions which permitat least the doubling of cells after 4 weeks. The term may be used todenote cultures of both undifferentiated pluripotent stem cells andcultures of differentiated cells derived from stem cells (either bydirected differentiation or by spontaneous differentiation).

“Long term” as used herein with regard to cell cultivation andexpansion, refers to the cultivation of SC for at least X weeks,preferably, for at least 6 weeks and more preferably, for at least 10weeks.

“Laminin-free culture system” refers to any culture system which has notbeen supplemented with laminin or laminin equivalent which being capableof providing culture conditions for the maintenance and/or expansion ofstem cells.

“Cell marker”, as used herein, refers to is any phenotypic feature of acell that can be used to characterize it or discriminate it from othercell types. A marker may be a protein (including secreted, cell surface,or internal proteins; either synthesized or taken up by the cell); anucleic acid (such as an mRNA, or enzymatically active nucleic acidmolecule) or a polysaccharide. Included are determinants of any suchcell components that are detectable by antibody, lectin, probe ornucleic acid amplification reaction that are specific for the cell typeof interest. The markers can also be identified by a biochemical orenzyme assay that depends on the function of the gene product.Associated with each marker is the gene that encodes the transcript, andthe events that lead to marker expression. A marker is said to bepreferentially expressed in an undifferentiated or differentiated cellpopulation, if it is expressed at a level that is at least 5 timeshigher (in terms of total gene product measured in an antibody or PCRassay) or 5 times more frequently (in terms of positive cells in thepopulation). Markers that are expressed 10, 100, or 10,000 times higheror more frequently are increasingly more preferred.

“Culture systems that supports differentiation into selected populationof somatic cells”, as used herein, refers to a variety of culturesystems known in the art to promote specific differentiation to aspecifically desired population of somatic cells. For example, a culturesystem that supports the directed differentiation of SCs into neuralprecursor cells may comprise a basic medium supplemented by FGF2 and/ornoggin, as described, for example by Itsykson, P., et al.¹⁴. Further,for example, a culture system that supports the directed differentiationinto dopaminergic neuronal cells will initially comprise the sameconditions supporting differentiation into neural precursor cells, thelatter directed into dopaminergic neuronal cells by the supplementationof the medium with at least one of sonic hedgehog (SHH), fibroblastgrowth factor (FGF), or a member of the Wnt family¹⁵.

“Culture conditions/systems that support spontaneous differentiationinto somatic cells”, as used herein, refers to any culture conditionsthat promoted spontaneous nonspecific differentiation of stem cells to amixture of somatic cells from any of the three embryonic germ layers:ectoderm; mesoderm; and endoderm. The medium in such culture conditionswill typically be without components known to be required for themaintenance of SCs in an undifferentiated (pluripotent) state. Suchcomponents typically include soluble factors typically added to mediafor maintenance of undifferentiated SCs. An example of a medium thatsupports spontaneous differentiation of SCs into somatic cells comprisesa basic medium of DMEM supplemented by FCS 20%,as described by Reubinoffet al.¹.

(If you agree with the above corrections we need to correct itthroughout the document)

In accordance with the broadest aspect, the present invention provides aculture system for expansion of SCs comprising a suspension ofundifferentiated SCs within basic medium and knockout serum replacement(KOSR). As indicated above, it was surprisingly found that thecombination of KOSR with a basic medium provided conditions suitable forthe expansion of pluripotent SCs. The SCs were maintained in anundifferentiated state for a long period, i.e. of at least 6 weeks.

The culture system in accordance with the invention comprise thesuspension wherein the SCs are in the form of free floatingundifferentiated SCs, free floating clusters of undifferentiated SCs orfree floating aggregates of undifferentiated SCs.

SCs can be obtained using well-known cell-culture methods. For example,hESC can be isolated from human blastocysts, morulas, cleavage stageembryos or blastomeres. Human blastocysts are typically obtained fromhuman preimplantation embryos, from in vitro fertilized (IVF) embryos orparthenogenetically activated oocytes. Alternatively, a single cellhuman embryo can be expanded to the cleavage stage, morula or blastocyststage. For the isolation of human ES cells from blastocysts, mostcommonly the zona pellucida is removed from the blastocyst and the innercell mass (ICM) is isolated by immunosurgery, in which the trophectodermcells are lysed and removed from the intact ICM by gentle pipetting. TheICM is then plated in a tissue culture flask containing the appropriatemedium which enables its outgrowth. Following 9 to 15 days, the ICMderived outgrowth is dissociated into clumps either by a mechanicaldissociation or by an enzymatic degradation and the cells are thenre-plated on a fresh tissue culture medium. Colonies demonstratingundifferentiated morphology are individually selected by micropipette,mechanically dissociated into clumps, and re-plated. Resulting ES cellsare then routinely split every 1-2 weeks. For further details on methodsof preparation human ES cells see Thomson et al. [U.S. Pat. No.5,843,780; Science 282:1145, 1998; Curr. Top. Dev. Biol. 38:133, 1998;Proc. Natl. Acad. Sci. USA 92: 7844, 1995]; as well as Bongso et al.[Hum Reprod 4: 706, 1989]; Gardner et al. [Fertil. Steril. 69:84, 1998];and Klimanskaya et al. [Nature. 446: 342, 2007].

Commercially available SCs can be also be used in accordance with theinvention. hESCs can be purchased from the NIH human embryonic stemcells registry. Non-limiting examples of commercially availableembryonic stem cell lines are BG01, BG02, BG03, BG04, CY12, CY30, CY92,CY10, TE03 and TE32.

Pluripotent SCs present at their surface, or express, biological markerswhich are used to identify pluripotent SCs as well as to verify that thecells in the culture are maintained in an undifferentiated state[Thomson J A et al. Embryonic Stem Cell Lines Derived from HumanBlastocysts Science 282(5391):1145-1147 (1998)]. A non-limiting list ofsuch cell markers comprise stage-specific embryonic antigens such asSSEA-3 and SSEA-4; antibodies to specific extracellular matrix moleculewhich are synthesized by undifferentiated pluripotent SC, such asTRA-1-60, TRA-1-81, and GCTM-2; elevated expression of alkalinephosphatase, which is associated with undifferentiated pluripotent SCs;and transcription factors unique to pluripotent SCs and which areessential for establishment and maintenance of undifferentiated SCs,such as OCT-4, Nanog and Genesis [Carpenter, M. K., Rosler, E., Rao, M.S., Characterization and Differentiation of Human Embryonic Stem Cells.Cloning and Stem Cells 5, 79-88, 2003].

Generally, the basic medium may be any basic medium known in the art. Ina preferred embodiment the basic medium is selected from Neurobasal™,Cellgro Stem Cell Growth Medium, KO-DMEM and X-Vivo 10. Most preferablythe present invention makes use of Neurobasal™ as the basic medium (i.e.the basic media consists essentially of Neurobasal™). Neurobasal™ isknown in the art of cell cultures [Brewer G J. Serum-free B27/Neurobasalmedium supports differential growth of neurons from the striatum,substantia nigra, septum, cerebral cortex, J Neurosci Res. 42(5):674-83,(1995)] and is commercially available [Gibco, Invitrogen cell culture,USA].

The culture system, e.g. Neurobasal TM supplemented with KOSR, may besupplemented by other components known to be used in culture systems,comprising, without being limited thereto, a member of FGF family. Inaccordance with one embodiment, the FGF member is, without being limitedthereto, FGF2.

The culture system may be further supplemented by an extracellularmatrix (ECM) component. In accordance with one embodiment, the ECM isselected from, without being limited thereto, fibronectin, laminin andgelatin.

The culture system may be further supplemented by an antibacterialagent. The antibacterial agent may be selected from, without beinglimited thereto, penicillin and streptomycin.

The culture system may be further supplemented by non-essential aminoacids (NEAA).

In addition, the culture system may be further supplemented by a TGFβsuperfamily factor. The TGFβ superfamily factor may be, without beinglimited thereto, activin A.

The culture system may be further supplemented by a neurotrophin.Neutrophins are known to play a role in assisting to promote thesurvival of SCs in culture. In accordance with an embodiment of theinvention, the neurotrophin is selected from, without being limitedthereto, BDNF, NT3, NT4.

The culture system may be further supplemented by nicotinamide (NA). Itis noted that NA may assist in preventing the differentiation of cellsinto extraembryonic lineages, maintaining them as undifferentiatedcells, assist in promoting the cells' survival and proliferation.

The culture system may be further supplemented by a bone morphogenicprotein (BMP) antagonist. It is noted that under culture conditions thatsupport undifferentiated proliferation of hESCs noggin (a BMPantagonist) prevents extraembryonic background differentiation of hESCs.While under conditions that promote differentiation, noggin is known toprevent the differentiation to non-neural lineages, favoring thedifferentiation to a neural fate. The BMP antagonist may be selectedfrom, without being limited thereto, noggin, chordin, or gremlin.

Further, the culture system may be supplemented by a serum free mediumsupplement. The serum free medium supplement is selected from, withoutbeing limited thereto, Nutridoma-CS or TCH™. Preferably the culturesystem is supplemented by Nutridoma-CS.

The culture system of the invention is preferably applicable for theexpansion in a suspension of embryonic SCs. A further preferredembodiment of the invention encompasses the expansion in a suspension ofhuman SCs.

A preferred embodiment of the invention comprises a culture system forthe expansion of hESCs in an undifferentiated state, the hESCs beingpluripontent stem cells. Most preferably, the invention provides aculture system for the expansion of hESCs, the culture system comprisinga suspension of such cells in a medium comprising Neurobasal™ and KOSR,the culture system permitting expansion of the cells in anundifferentiated pluripotent state. In a preferred embodiment, theinvention provides a culture system for the expansion of hESCs, theculture system comprising a suspension of such cells in a basic mediumconsisting of Neurobasal™ and the culture system comprising KOSR, theculture system permitting expansion of the cells in an undifferentiatedpluripotent state.

The results presented hereinbelow also show that the culture system maybe free of laminin. Laminin has been shown in culture to stimulateneurite outgrowth, promote cell attachment, chemotaxis, and celldifferentiation. The present invention has found that when supplementingthe basic media with a serum free medium supplement, e.g. Neutridoma CS,not only there is no need to add laminin but also, an increase number ofcells was observed after a long term of cultivation in suspension, ascompared to the number of cells outgrown in the presence of laminin.

The culture system of the invention is preferably suitable for long termexpansion of SCs. In accordance with one embodiment, long term refers toat least six weeks of cultivation and cell expansion. During this term,the SCs exhibit cell markers which confirm that the SCs are essentiallymaintained in an undifferentiated pluripotent state.

It should be well appreciated by those versed in the art that a culturesystem in accordance with the invention, i.e. a culture system for theexpansion of SCs in a suspension, wherein the SCs are maintained for along period of time in an undifferentiated, pluripotent state, may be ofsignificant benefit for large scale propagation of SCs in bioreactors.As well appreciated by those versed in the art, it is advantageous toprovide conditions that allow for large scale production of standardizedhomogeneous undifferentiated hESCs. The advantages of suspension culturebioreactors were also described above, in particular for large scaleexpansion.

The invention also provides a method of expanding SCs in anundifferentiated state, the method comprising incubatingundifferentiated SCs in suspension within a culture system comprisingbasic medium and knockout serum replacement (KOSR). Preferably, themethod comprises incubating undifferentiated SCs in suspension within aculture system comprising basic medium consisting of Neurobasal™ andfurther comprising knockout serum replacement (KOSR).

The method of the invention also comprises the step of obtaining SCsfrom SC colonies cultivated on a feeder layer or in a feeder freeadherent culture system. The SCs may be obtained by dissociation of thecells from the culture system, e.g. by the aid of suitable agents (e.g.collagenase IV), trituration and transferring the dissociated SCs intothe culture system of the invention to form a suspension of SCs, thelatter being in the form of free floating cells, free floating clustersof cells or free floating aggregates of cells.

The method may also comprise one or more media refreshment (i.e. thereplacement of at least 50% of the culture system). It is appreciatedthat by said media refreshment, dead cells and their fragments aregradually removed. Culture media may be refreshed at least every 2-3days, and most preferably at least every 2 days. The media refreshmentmay include the replacement of a portion of the basic media only, aswell as the replacement of a portion of the basic media including one ormore of its components. Further, it is appreciated that the method maycomprise different media replacements, e.g. at times only thereplacement of the basic medium, and at other time points, thereplacement of the basic medium comprising one or more of thesupplements.

It is also appreciated that as a result of cells expansion, the SCs mayproliferate into big clusters. Thus, the method of the invention mayalso comprise one or more SCs manipulations so as to disaggregate thebig clusters of cells resulting from their overgrowth. According to oneembodiment, the overgrowth of the cells in clusters is prevented bytrituration. The essentially disaggregated cells may then be transferredto suitable tissue culture carriers (e.g. dishes, culture tubes, culturebioreactors, etc.) for continued expansion. Overgrowth of clusters maybe prevented by other means such as chopping the clusters or the use ofincreased shearing forces of bioreactor systems or any other methodknown in the art.

It has been found that the undifferentiated and pluripotent SCs obtainedusing the culture system of the invention may be, at any stage ofexpansion, induced to differentiate into a variety of somatic cells fromthe three embryonic major lineages; endoderm, ectoderm and mesoderm.

Depending on the type of culture system used for the induction ofdifferentiation, the nature of the resulting population may be a prioridetermined.

In accordance with one embodiment, the undifferentiated and pluripotentSCs are induced to differentiate into a specific and pre-selected fate.In other words, the undifferentiated and pluripotent SCs are cultivatedin a culture system that directs differentiation to a specific somaticcells, thereby providing a population of cells highly enriched for aspecific cell type or a pure population of cells of a single type. Avariety of single type somatic cell populations may be derived fromundifferentiated and pluripotent SCs and those versed in the art willknow how to select the medium components and establish the culturesystem which direct the specific differentiation of the latter to thedesired population of somatic cells.

For example, directing differentiation of undifferentiated andpluripotent SCs to neural precursor cells may be obtained by cultivatingthe SCs in a culture system comprising DMEM/F12 medium (Gibco)supplemented with B27 (1%, Gibco) (DMEM/F12/B27 medium), FGF-2 (20ng/ml) and noggin (750 ng/m, R&D Systems, Inc., Minneapolis, Minn.) asexemplified hereinbelow and also by Itsykson, P., et al.¹⁴) or byReubinoff et al.⁽²⁴⁾.

Further, for example, directing differentiation of SCs to midbraindopamineric neuronal cells may be obtained by first by inducingdifferentiation into neural precursor cells, such as described above,followed by cultivation in a basic medium, such as DMEM/F12/B27 mediumsupplemented with at least one of fibroblast growth factor 8 (FGF8) andsonic hedgehog (SHH), a member of the Wnt family, as exemplified belowand also described by Yan, Y. et al.¹⁵. Co-culture with cells thatpromote midbrain differentiation such as the PA6 stromal cells, ormidbrain astrocytes may be also used.

In accordance with another embodiment, the SCs may be induced forspontaneous non-specific differentiation of somatic cells. This may beachieved, for example, by the use of a culture media that is free ofsoluble factors and/or ECM components typically used for maintaining SCsin undifferentiated state, the exclusion thereof from the culture mediais known to promote spontaneous differentiation of SCs, as alsodescribed by Itsykson, P., et al.⁽¹⁴⁾ Reubinoff et al [Nat Biotechnol18, 399-404 (2000)].

In accordance with this embodiment, within the culture system theundifferentiated pluripotent SCs (in suspension) are induced to formembryoid bodies (EBs). The said EBs are disaggregated and further platedand cultured in the same medium of the EBs. It is noted that theproduction of a mixture of non-specific somatic cells was exhibited byexpression of human muscle actin, indicating the presence of mesodermalcells in the cell culture; or by expression of Sox-17, indicating thepresence of endodermal cells in the cell culture.

Methods other than spontaneous differentiation within EBs may be used toderive somatic cells for example differentiation in flat culture; andother culture systems may be used to promote differentiation towardsspecific cell types^((26,27))

It is noted that the undifferentiated pluripotent SCs of the inventionmay also be used for the production of Teratoma tumors. This may beachieved by injection of the pluripotent SCs into an animal.

As indicated above, the culture system of the invention is especiallysuitable for large scale production of SCs. Thus, it should beappreciated that the methods of the invention for the production ofspecific as well as non-specific somatic cells, making use of the SCsproduced using the culture system of the invention, are also suitablefor large scale production of such somatic cells. In other words, thepresent invention provides methods for the large scale production of SCsas well as for the large scale production of somatic cells derivedtherefrom (either by spontaneous or directed differentiation).

It is appreciated that certain features of the invention, which are, forclarity, described in the context of separate embodiments, may also beprovided in combination in a single embodiment. Conversely, variousfeatures of the invention, which are, for brevity, described in thecontext of a single embodiment, may also be provided separately or inany suitable sub-combination.

Although the invention has been described in conjunction with specificembodiments thereof, it is evident that many alternatives, modificationsand variations will be apparent to those skilled in the art.

All publications, patents and patent applications mentioned in thisspecification are herein incorporated in their entirety by referenceinto the specification.

The invention will now be described with reference to the followingnon-limiting examples.

Some Exemplary Embodiments

Materials and Methods

Human Feeders and Preparation of Feeder Layers

Foreskin cells from an established line were used as feeders aspreviously described (Banin E, Obolensky A, Idelson M, Hemo I,Reinhardtz E, Pikarsky E, Ben-Hur T, Reubinoff B. Retinal incorporationand differentiation of neural precursors derived from human embryonicstem cells. Stem Cells 2006; 24(2): 246-57). Briefly, the foreskinfibroblasts were cultured in DMEM (Gibco, Gaithersburg, Md.)supplemented with 10% Fetal Calf Serum (FCS) (Biological Industries,Beit Haemek, Israel). They were passaged by trypsin (Gibco) digestion.For the preparation of feeder layers 3×10⁵ cells were plated per well ofa six well plate (Corning, N.Y., USA), precoated with 0.1% gelatin(Sigma, St. Louis, Mo.). Mitotic inactivation of the feeders was carriedout prior to plating by incubating them 2.5 hours with Mitomycin-C 5μg/ml (Kyowa, Tokyo).

hESC Culture System

hESCs were cultured on the human feeder layers in KO medium (KOM)consisting of 85% KO-DMEM, 15% KOSR, 1 mM glutamine, 0.1 mMβ-mercaptoethanol, 1% nonessential amino acids, 50 units/ml penicillin,50 μg/ml streptomycin, (Gibco, Gaithersburg, Md.) and 4 ng/ml FGF-2 (R&DSystems, Inc., Minneapolis, Minn.). hESCs were weekly passaged withCa/Mg⁺⁺-free PBS supplemented with 0.05% EDTA (Biological Industries,Beit Haemek, Israel) or type IV collagenase (1 mg/ml; Gibco) and platedonto fresh feeder layer.

Suspension Culture of hESC

hESC colonies that were cultivated on human feeders in the KO culturesystem described above and in WO 2006/070370, the entire contents ofwhich is specifically incorporated herein by reference, were firstlydissociated with the aid of collagenase IV (1 mg/ml, 2-3 hours at 37°).Cell dissociation was promoted by agitation of the culture plate. Thecell clusters obtained were resuspended in fresh NBSR medium(Neurobasal™, 14% KOSR, glutamine 2 mM, 50 units/ml penicillin, 50 μg/mlstreptomycin, 1% nonessential amino acids; Gibco), or where indicated infresh NBN2 medium (Neurobasal™, N2 supplement 1:100, glutamine 2 mM, 50units/ml penicillin, 50 μg/ml streptomycin). Both media weresupplemented with FGF-2 20 ng/ml, activin 25 ng/ml, fibronectin andlaminin 5 μg/ml each, gelatin 0.001%, and BDNF, NT3 and NT4, 10 ng/mleach.

When indicated, cell clusters were resuspended in NBSR mediumsupplemented with 1× Nutridoma-CS (Roche, Germany), FGF-2 20 ng/ml,activin 25-50 ng/ml, fibronectin 5 μg/ml, and gelatin 0.001%, with andwithout laminin 5 μg/ml.

The suspension was either strained though 30-50 micron mesh to removebig clumps, or triturated by pipetting to disaggregate big clumps andtransferred into tissue culture dishes (Costar®, Corning Inc., Corning,N.Y.) at a density of 0.7-1.2×10⁶ cells/ml. Dead cells and theirfragments were gradually removed during media refreshment every twodays. The cells proliferated as free-floating tiny transparent clustersof 20-50 cells. Aggregation and overgrowth of clusters was prevented bytrituration with a 1000 μl pipettor tip as required.

Characterization of hESC Grown in Suspension by FACS Analysis

For characterization of the cells within the small free-floatingaggregates, hESCs were dissociated with a solution of 2.25 mM EDTA with0.06% trypsin, for 7-10 min at 37°, followed by gentle trituration, toobtain a single-cell suspension solution.

The hESC were then washed with FACS media consisting of PBS supplementedwith 1% BSA and 0.05% sodium azide. The single-cell suspension wasstained with anti-SSEA4 (1:100, mouse monoclonal IgG3, DevelopmentalStudies Hybridoma Bank (DHSB), Iowa City, Iowa), anti-Tra-1-60 (1:100,monoclonal mouse IgM, Chemicon International), and anti-Tra-1-81 (1:100,monoclonal mouse IgM, Chemicon International). Control hESCs werestained with their respective isotype control antibodies. Primaryantibodies were detected using fluorescein isothiocyanate (FITC)-labeledgoat anti-mouse Ig (1:100, Dako). Propidium iodide (PI) was added (finalconcentration of 4 μg/ml) for better gating of viable cells. FACSanalysis was performed using the FACSCalibur system (Becton-Dickinson,San Jose, Calif.).

Replating and Monolayer Culture of hESCs Cultivated in Suspension

Floating aggregates of hESCs were triturated with a 1000 μl pipettortip. Tiny clusters that were obtained were plated on fresh feeders andcultured in KOSR medium (as above) supplemented with 4 ng/ml FGF2. After1 week, colonies with typical morphological characteristics of thecolonies of undifferentiated hESCs, developed on the feeders. Thesecolonies could be passaged routinely as described before. Alkalinephosphatase activity of the cells within the plated colonies wasdemonstrated using the Alkaline Phosphatase Substrate kit I by VectorLaboratories (Burlingame, Calif.) according to the manufacturer'sinstructions.

EB Formation

The clusters of undifferentiated hESCs were transferred and cultured for3-4 weeks in DMEM (Gibco), supplemented with 20% FBS (BiologicalIndustries, Beit Haemek), 1 mM L-glutamine, 0.1 mM β-mercaptoethanol, 1%non-essential amino acid stock, 50 units/ml penicillin, 50 μg/mlstreptomycin (all from Gibco Invitrogen Corporation products, USA).

Induction of Somatic Differentiation and Immunohistochemistry

Clusters of undifferentiated hESCs were transferred and cultured for 2-3weeks in suspension within bacteriological dishes precoated with 0.1%low melting temperature agarose in DMEM/F12 medium (Gibco) supplementedwith B27 (1%, Gibco) (DMEM/F12/B27 medium), FGF-2 (20 ng/ml) and noggin(750 ng/m, R&D Systems, Inc., Minneapolis, Minn.).

Clumps of neural precursors were triturated to small clusters and platedon poly-D-lysine (30-70 kDa, 10 μg/ml; Sigma, St. Louis, Mo.) andlaminin-coated (4 μg/ml; Sigma) glass coverslips and cultured for anadditional week with DMEM/F12/B27 medium in the absence of growthfactors.

In addition, to promote differentiation to midbrain dopaminergicneurons, clumps of neural precursors were triturated to small clustersand plated on poly-D-lysine and laminin-coated glass coverslips (asabove) and cultured for 2 weeks with DMEM/F12/B27 medium supplementedwith FGF8 and SHH.

EBs were dissociated with the aid of trypsin (0.025%, 3 mM EDTA in PBS)digestion, and plated on poly-D-lysine and laminin pre-coated glasscoverslips (as above) and cultured for additional 1-2 weeks in theculture medium used for induction of differentiation of EBs.

Differentiated cells within the outgrowth were fixed with 4%paraformaldehyde for 20 minutes at room temperature. Cell membranes werepermeabilized with 0.2% Triton X100 (Sigma) in PBS for 5 minutes forimmunostaining with anti-intracellular marker antibodies. Neuralprecursors were incubated with the following primary antibodies:anti-β-III-tubulin (mouse monoclonal IgG2b, 1:2000, Sigma), anti-rabbitTH (1:200, Pel Freeze), and anti-mouse EN-1 (1:100, DevelopmentalStudies Hybridoma Bank (DHSB), Iowa City, Iowa). Mesodermaldifferentiation within EBs' outgrowth was detected with the antibodyagainst human muscle actin (1:50, DAKO). Endodermal differentiationwithin EBs' outgrowth was detected with the antibody against Sox-17(1:50, R&D Systems Inc.). Primary antibody localization was performed byusing fluorescein isothiocyanate (FITC)-conjugated goat anti-rabbitimmunoglobulins (Dako, 1:20-50), or goat anti-mouse immunoglobulinconjugated with Cy3 (1:500 Jackson ImmunoResearch Laboratories).

Teratoma Formation in NOD/SCID Mice

Clumps of hESCs that were propagated for 7 weeks in suspension wereinjected into the testis of six weeks old NOD/SCID mice (Harlan,Jerusalem, Ill.) (30-40 clumps per testis). Eight to twelve weeks later,the resulting tumors were removed, embedded in paraffin and sectionswere stained with H&E.

Results

NBSR Suspension Culture System for the Propagation of hESC in Bulk

To develop suspension cultures, hESC colonies that were cultivated onhuman feeders in the KO culture system were dissociated with the aid oftype IV collagenase. The cells/cell clusters that were obtained werere-suspended within fresh NBSR medium, supplemented with FGFs (FGF-2 20ng/ml).

Further supplementation of the medium with one or more of the followingcomponents increased the survival/proliferation and preventeddifferentiation of the cells:

-   -   TGFβ superfamily factors (e.g., activin 25-50 ng/ml)    -   ECM components (e.g., laminin 5 μg/ml, fibronectin 5 μg/ml,        gelatin 0.001%)    -   Neurotrophins (e.g., NT4, NT4, BDNF 10 ng/ml each)

The cells were transferred to suspension at a density of ˜0.7-1.2×10⁶cells/ml. Dead/fragmented cells were gradually removed during mediumrefreshment. The cells proliferate as free-floating tiny transparentclusters of 20-50 cells without any morphological signs ofdifferentiation (FIG. 2). Aggregation and overgrowth of the transparentclusters were prevented by trituration through a 1000 μl pipette tip asrequired. The hESCs grown in a suspension comprising Neurobasal™ mediumsupplemented with KOSR (NBSR) were sub-cultured by mechanicaldisaggregation.

The cells expressed SSEA4, TRA1-60, and TRA1-81 (markers ofpluripotency), and did not express markers of somatic differentiationsuch as the neural marker PSA-NCAM. Specifically, after 3 weeks ofcultivation in suspension under these culture conditions, >90% of thecells expressed SSEA4, TRA1-60, and TRA1-81 but not PSA-NCAM (FIGS.3A-3E).

When re-plated on human feeders, after 3 and 7 weeks of suspensionculture, the cells gave rise to monolayer colonies with the morphologyof undifferentiated hESCs (FIG. 4A). The stem cells within thesecolonies expressed alkaline phosphatase (a marker used to identify stemcells; FIG. 4B).

It was concluded that efficient propagation of undifferentiated hESC insuspension is achievable using the unique combination of Neurobasal™medium as the basic medium of the culture system, supplemented with KOSR(NBSR).

Suspension in NBSR was shown to be more effective than suspension inNBN2 (Neurobasal™ supplemented with N2) for the expansion of hESCswithout differentiation. Specifically, after 3 weeks, the total numberof cells in each of the cell cultures (NBSR vs. NBN2) was determinedusing trypan blue to include only live cells and hESCs propagated inNBSR reached a population of approximately double that of NBN2-culturedhESCs (FIG. 1).

NBSR medium that was supplemented with Nutridoma-CS, FGF-2, activin, ECMcomponents (fibronectin, gelatin and laminin), and neutrophins couldeffectively support long-term cultivation of undifferentiated hESCs. Thepercentage of hESCs expressing markers of pluripotent stem cells (SSEA4,TRA1-60, and TRA1-81) was high (>95%) and stable after 3.5 weeks and 6.5weeks of suspension culture (FIG. 5) as well as after 7 and 10 weeks(FIG. 12).

Furthermore, NBSR medium supplemented with Nutridoma-CS did not requirethe addition of laminin. Using the same supplementation as describedabove, however, with or without laminin showed that the percentage ofundifferentiated cells cultured in NBSR/Nutridoma-CS was notsignificantly different in laminin-positive cultures (+LAM) versuscultures in which laminin was not added (−LAM) (FIG. 6). All the moreand quite surprisingly, in the absence of laminin, an increased numberof cells was observed after long-term cultivation (6.5 weeks) in theabove described NBSR/Nutridoma-CS culture suspension (FIG. 7).

Induced Differentiation of hESCs Into Somatic Cells In Vitro

Undifferentiated hESCs that were cultivated in suspension within NBSRgave rise upon differentiation to somatic progeny.

To induce neural differentiation the clusters were transferred andcultured for 2 weeks in DMEM/F12 supplemented with B27 (1%), FGF-2 (20ng/ml) and noggin (750 ng/ml). These culture conditions had beenpreviously demonstrated to be highly efficient for induction of neuraldifferentiation of hESCs⁽¹⁴⁾. The clusters were then plated onlaminin-coated slides and cultured for a week in the same medium in theabsence of growth factors. Cells with morphological characteristics ofneurons (FIG. 8A) gradually migrated from the clusters. Immunostainingdemonstrated that these cells expressed the neuronal markerβ-III-tubulin (FIG. 8B). When the clusters were plated on laminin coatedslides for 2 weeks of differentiation in the presence of FGF8 and SHH(which promote differentiation toward a midbrain dopaminergic neuronalfate), multiple neurons co-expressing enlgraid-1 (EN-1) and tyrosinehydroxylase (TH) were observed (FIG. 9). The co-expression of thesemarkers is characteristic of midbrain dopaminergic neurons¹⁵. The arrowshows a cell which co-expresses EN-1 and TH. The arrow is hard to see onthe B/W figure. On the B/W scale, TH is indicated by light gray, andEN-1 is indicated by darker gray.

To induce mesodermal and endodermal differentiation the clusters weretransferred to DMEM supplemented with 20% FBS where they formed embryoidbodies (EBs). After 3-4 weeks of differentiation the EBs weredissociated, plated and cultured for 1-2 weeks on laminin coatedcoverslips. Immunostaining showed differentiated cells expressing muscleactin (mesoderm; FIG. 10), and Sox 17 (endoderm; FIG. 11).

To further demonstrate that the hESCs retained their pluripotentpotential after cultivation in suspension, clusters of hESCs that werecultured in suspension for 7 weeks were injected into the testis ofNOD/SCID mice. Teratoma tumors were removed after 8 weeks andhistological analysis of the tumors demonstrated differentiated progenyof the three embryonic germ layers (FIGS. 13A-13C). Formation ofteratoma is a key feature of hESCs and is an important evidence that thecells that grow in suspension are indeed pluripotent cells.

1. A method of expanding SCs in an undifferentiated state, the methodcomprising incubating undifferentiated SCs in suspension within aculture system comprising basic medium and knockout serum replacement(KOSR).
 2. The method of claim 1, wherein said undifferentiated SCs arederived from SC colonies cultivated on a feeder layer or in a feederfree adherent culture system.
 3. The method of claim 1, wherein theundifferentiated SCs in said suspension are in form of free floatingcells, free floating clusters of cells or free floating aggregates ofcells.
 4. The method of claim 1, wherein said basic medium isNeurobasal™.
 5. The method of claim 1, wherein said culture systemcomprises one or more of the following: a member of FGF family; aextracellular matrix (ECM) component; an antibacterial agent;nonessential amino acids; a TGFβ superfamily factor; a neurotrophin;nicotinamide (NA); a bone morphogenic protein (BMP) antagonist; or aserum free medium supplement.
 6. The method of claim 5, wherein said ECMis selected from fibronectin and gelatin; said antibacterial agent isselected from penicillin and streptomycin; said TGFβ superfamily factoris selected from activin A; said BMP antagonist is selected from noggin,chordin; and said serum free medium supplement is selected fromNutridoma-CS or TCH™.
 7. The method of claim 1, for expandingpluripotent hESCs in an undifferentiated state.
 8. The method of claim1, for large-scale expansion and/or long term cultivation ofundifferentiated SCs in suspension.
 9. A method for directingdifferentiation of SCs into a selected population of somatic cells froma culture system of SCs in suspension, the method comprising: (a)providing a suspension of undifferentiated SCs comprising a basic mediumand knockout serum replacement (KOSR); and (b) incubating saidundifferentiated SCs in culture system that support directeddifferentiation of SCs into the selected population of somatic cells.10. The method of claim 9, wherein said SCs are pluripotent hESC. 11.The method of claim 9, wherein the somatic cells are selected frommesodermal, endodermal or ectodermal cells.
 12. The method of claim 9,wherein said selected population of somatic cells consists essentiallyof a single population of differentiated somatic cells.
 13. The methodof claim 9, wherein said population of somatic cells consistsessentially of neural precursor cells.
 14. The method of claim 13,wherein the culture system comprises a basic media, FGF2 and/or noggin.15. The method of claim 12, wherein said selected population of somaticcells consists essentially of dopaminergic neuronal cells.
 16. Themethod of claim 15, comprising directing differentiation of SCs intoneural precursor cells and incubating said neural precursor cells in aculture system supporting differentiation of neural precursor cells intodopaminergic neuronal cells.
 17. The method of claim 16, wherein saidculture system supporting differentiation of neural precursor cells intodopaminergic neuronal cells comprises at least one of sonic hedgehog(SHH), an fibroblast growth factor (FGF), or a member of the Wnt family.18. The method of claim 9, wherein said SCs are pluripotent hESC.
 19. Amethod for promoting spontaneous differentiation of SCs into somaticcells, the method comprising: (a) providing a suspension ofundifferentiated SCs in a culture system comprising a basic medium andknockout serum replacement (KOSR); (b) incubating said undifferentiatedSCs in culture system that support spontaneous differentiation of SCsinto the somatic cells.
 20. The method of claim 19, wherein said somaticcells comprise a mixture or any one of mesodermal cells, ectodermalcells, endodermal cells and combination of same.
 21. The method of claim19, wherein said
 22. A culture system for expansion of stem cells (SCs)comprising a suspension of undifferentiated stem cells within basicmedium and knockout serum replacement (KOSR).
 23. The culture system ofclaim 22 wherein said suspension of stem cells comprises free floatingcells, free floating clusters of cells or free floating aggregates ofcells.
 24. The culture system of claim 22, wherein said the basic mediumis Neurobasal™.
 25. The culture system of claim 22, comprising one ormore of the following: a member of FGF family; an extracellular matrix(ECM) component; an antibacterial agent; non-essential amino acids aTGFβ superfamily factor; a neurotrophin; nicotinamide (NA); a bonemorphogenic protein (BMP) antagonist; or a serum free medium supplement.26. The culture system of claim 25, wherein said FGF member is FGF-2,ECM is selected from fibronectin, laminin and gelatin; saidantibacterial agent is selected from penicillin and streptomycin; saidTGFβ superfamily factor is activin A; said BMP antagonist is selectedfrom noggin, chordin, or gremlin, said neurotrophin is selected fromBDNF, NT3, NT4; and said a serum free medium supplement is selected fromNutridoma-CS or TCH™.
 27. The culture system of claim 22, wherein saidSCs are embryonic SCs.
 28. The culture system of claim 22, wherein saidSCs are pluripotent, human embryonic SCs (hESCs).
 29. The culture systemof claim 22, being free of laminin.
 30. The culture system of claim 22,wherein said SCs are expanded for long term and/or in large scale in anundifferentiated state.
 31. Use of KOSR for the preparation of a culturesystem for the maintenance of SCs in an undifferentiated pluripotentstate.
 32. Use of claim 31, wherein said SCs are pluripotent hESCs. 33.Use of claim 31, for large scale and/or long term expansion of said SCsin a bioreactor.
 34. The method of any one of claims 1 for large scaleexpansion and/or long term expansion of pluripotent hESCs in abioreactor.
 35. The method of claims 9 for large scale differentiationof pluripotent hESCs in a bioreactor.